Tissue cGMP Research Articles

Vasorelaxation induced by the new nitric oxide donor cis-[Ru(Cl)(bpy) 2(NO)](PF 6) is due to activation of K Ca by a cGMP-dependent pathway

We investigated the effects of selective K + channel blockers and guanylyl cyclase inhibitor on the rat aorta relaxation induced by the new NO donor cis-[Ru(Cl)(bpy) 2(NO)](PF 6) (RUNOCL), following endothelium removal. NO release from RUNOCL was obtained by photo-induction using a visible light system λ > 380 nm. RUNOCL induced relaxation of phenylephrine contracted aortic rings under light with the maximum effect (ME) of 101.2 ± 3.7% and pD 2: 6.62 ± 0.16 ( n = 7), but not in the absence of light. Relaxation stimulated with RUNOCL was also studied on 60 mM of KCl-contracted arteries or after incubation with the non-selective K + channel blocker (1 mM TEA) or the selective K + channel blockers (3 μM glibenclamide (K ATP), 1 mM 4-aminopyridine (K V, 4-AP), 1 μM apamin (SK Ca-APA) or 0.1 μM iberiotoxin (BK Ca IBTX). Relaxation induced by RUNOCL was lower in KCl-contracted aortic rings with ME of 68.6 ± 10.0% and pD 2: 3.92 ± 0.60 ( n = 4). As compared to Phe-contracted arteries the potency of RUNOCL in inducing rat aorta relaxation was reduced by K + channel blockers as demonstrated by the pD 2 values from 6.62 ± 0.16 ( n = 7) (control) to (TEA: 5.32 ± 0.108, n = 5; IBTX: 5.63 ± 0.02 ( n = 5), APA: 5.73 ± 0.13 ( n = 5)). But the ME was reduced only by IBTX (60.7 ± 3.4%). 4-AP and glibenclamide had no effect on the relaxation induced by RUNOCL. The aortic tissue cGMP content increased with RUNOCL under light irradiation from 63.13 ± 0.45 fmol/μg to 70.56 ± 4.64 fmol/μg of protein ( n = 4) and the inhibition of guanylyl cyclase with ODQ reduced the ME: 30.1 ± 1.6% and pD 2: 6.35 ± 0.05 ( n = 4). Our results suggest that the NO released by photo-induction from RUNOCL induces rat aorta relaxation by activation of K Ca by a cGMP-dependent pathway.

Oral phosphodiesterase-5 inhibitors: effect of heme oxygenase inhibition on cGMP signalling in rat cavernous tissue

This work postulated that heme oxygenase (HO) is partly responsible for controlling phosphodiesterase-5 inhibitor actions by modulating cyclic guanosine monophosphate (cGMP) cavernous tissue levels. Five hundred and four male Sprague-Dawley rats, divided into five groups, were investigated. Group 1 (n=72) included controls, group 2 (n=72) received sildenafil citrate (Viagra) orally, group 3 (n=72) received vardenafil hydrochloride (Levitra), group 4 (n=72) received tadalafil (Cialis). Group 5 (n=216), subdivided into three subgroups (A, B and C, 72 each), received the same dose of each drug with the HO inhibitor, Zn protoporphyrin. Eight rats from each group/subgroup were killed at 0.5, 1, 2, 3, 4, 6, 18, 24 and 36 h when cGMP levels in the cavernous tissues were estimated. Cavernous tissue cGMP levels increased significantly in sildenafil, vardenafil and tadalafil-treated rats compared to the controls with significant decreases after HO inhibition. It is concluded that the effects of these PDE-5 inhibitors in rat cavernous tissue are partly mediated through HO activity via the cGMP signalling pathway.

Reduction in soluble guanylyl cyclase-specific activity following prolonged treatment of porcine pulmonary artery with nitric oxide

In a newly characterized cultured porcine pulmonary artery (PA) preparation, 24-h treatment with the nitric oxide (NO) donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) decreased the response to acutely applied DETA-NO compared with 24-h control (-log EC(50) 6.55 +/- 0.12 and 5.02 +/- 0.21, respectively). Treatment of PA with the cell-permeable superoxide dismutase mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride, did not change NO responsiveness in either freshly prepared or 24-h DETA-NO-treated PA. cGMP and cAMP phosphodiesterase activities were approximately equal in PA. Twenty-four-hour DETA-NO treatment did not change either cGMP or cAMP phosphodiesterase activities. Twenty-four hours in culture had no significant effect on soluble guanylyl cyclase (sGC) subunit mRNA expression, but 24-h DETA-NO treatment significantly decreased the expression of both sGCalpha(1) and sGCbeta(1). sGCbeta(1) protein expression was 42 +/- 4 ng/mg soluble protein. Twenty-four hours in culture without and with DETA-NO reduced sGCbeta(1) protein expression (36 +/- 3 and 31 +/- 3 ng/mg soluble protein, respectively, P < 0.025). Basal tissue cGMP [(cGMP)(i)] was significantly increased, and NO-induced (cGMP)(i) was significantly decreased by 24-h DETA-NO treatment. (cGMP)(i) normalized to the amount of sGC protein expressed in PA was significantly lower in PA treated for 24 h with DETA-NO compared with both freshly isolated and 24-h cultured PA. We conclude that prolonged NO treatment induces decreased acute NO responsiveness in part by decreasing both sGC expression and sGC-specific activity.

Sildenafil reduces L‐NAME‐induced severe hypertension and worsening of myocardial ischaemia–reperfusion damage in the rat

Phosphodiesterase-5 inhibitors are beneficial in pulmonary hypertension and congestive heart failure, the two conditions associated with coronary heart disease and ischaemia. We investigated whether sildenafil counteracts the cardiovascular alterations induced by N -nitro-L-arginine methyl ester (L-NAME) in the rat. Sildenafil was given orally to rats at doses of 0.37, 0.75 or 1.5 mg kg-1day-1 for four weeks, either alone or with L-NAME (35-40 mg kg-1 day-1 in the drinking water). Systolic blood pressure and urinary parameters (6-keto-prostaglandin F1alpha, thromboxane B2, 8-isoprostane-prostaglandin F2 and nitrite/nitrate) were measured in conscious rats. Isolated hearts were subjected to low flow ischaemia-reperfusion, and myocardial levels of guanosine 3', 5'cyclic monophosphate (cGMP) were determined. Endothelial vascular dysfunction was examined in aortic rings. Sildenafil dose-dependently prevented the rise in systolic blood pressure in L-NAME-treated rats. This activity was associated with a normalization of urinary 8-isoprostane-prostaglandin F2alpha and other biochemical parameters. In perfused hearts, the post-ischaemic ventricular dysfunction was worse in preparations from L-NAME-treated rats than in controls. Sildenafil dose-dependently reduced this effect, and creatine kinase and lactate dehydrogenase release were lower too. cGMP levels, which were low in myocardial tissue from L-NAME-treated rats, were restored by sildenafil. In noradrenaline-precontracted aortic rings from L-NAME-treated rats acetylcholine lost its vasorelaxant effect, and sildenafil restored it. In a rat model of chronic nitric oxide deprivation, where hypertension and aggravation of post-ischaemic ventricular dysfunction are associated with loss of vascular endothelium-relaxant function, sildenafil provided significant cardiovascular protection, primarily by maintaining tissue cGMP levels.

558: Human atrial natriuretic peptide ameliorates warm ischemia-reperfusion injury of the lung

Purpose: Atrial natriuretic peptide (ANP), a hormone synthesized by cardiac atria, possesses potent diuretic and natriuretic properties and a relaxant action on smooth muscle. These effects have been ascribed to the activation of guanylate cyclase receptors, increasing tissue cGMP levels. We hypothesized that carperitide, synthetic ANP, administered on reperfusion after warm ischemia would protect reperfused lungs.

Nicorandil Elevates Tissue cGMP Levels in a Nitric-Oxide-Independent Manner

The K+ channel opener nicorandil is a hybrid compound that contains nitrate in its structure. It has been reported that nicorandil can relax vascular tissue in vitro via a mechanism that involves activation of KATP channels and stimulation of soluble guanylyl cyclase. However, it is not known whether the increase of cGMP levels occurs through an elevation of nitric oxide (NO). The aim of the present study was to determine whether NO release was a direct effect of nicorandil. We reported here that nicorandil did not generate NO using ozone chemiluminescence detection methods in human or rat liver microsomes (P450-rich fractions) with addition of NADPH. However, nicorandil elevated cGMP levels in rat liver, aorta, and human coronary smooth muscle cells in vitro. The elevation was not inhibited by the NO trapping agent carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO). These results suggest that nicorandil elevates cGMP without NO generation.

ENDOTOXIN‐INDUCED ACUTE LUNG INJURY IS ENHANCED IN RATS WITH SPONTANEOUS HYPERTENSION

1. Acute lung injury (ALI), or acute respiratory distress syndrome, is a major cause of mortality in endotoxaemia. The present study tested whether the endotoxaemia-induced changes and associated ALI were enhanced in rats with established hypertension and to examine the possible mechanisms involved. 2. Fifty spontaneously hypertensive rats (SHR) and the same number of normotensive Wistar Kyoto (WKY) rats, aged 12-15 weeks, were used. The experiments were performed in conscious, unanaesthetized rats. Endotoxaemia was produced by intravenous lipopolysaccharide (LPS; 10 mg/kg). N(G)-Nitro-L-arginine methyl ester (L-NAME; 10 mg/kg, i.v.), L-N(6)-(1-iminoethyl)-lysine (L-Nil; 5 mg/kg, i.v.) and 3-morpholinosydnonimine (SIN-1; 5 mg/kg, i.v.) were given 5 min before LPS to observe the effects of nitric oxide synthase (NOS) inhibition and nitric oxide (NO) donation. 3. We monitored arterial pressure and heart rate and evaluated ALI by determining the lung weight/bodyweight ratio, lung weight gain, leakage of Evans blue dye, the protein concentration in bronchoalveolar lavage and histopathological examination. Plasma nitrate/nitrite, methyl guanidine, pro-inflammatory cytokines, including tumour necrosis factor-alpha and interleukin-1beta, and lung tissue cGMP were determined. Expression of mRNA for inducible and endothelial NOS was examined using reverse transcription-polymerase chain reaction. 4. Lipopolysaccharide caused systemic hypotension, ALI and increases in plasma nitrate/nitrite, methyl guanidine, pro-inflammatory cytokines and lung cGMP content. The LPS-induced changes were greater in SHR than in WKY rats. Pretreatment with L-NAME or L-Nil attenuated, whereas the NO donor SIN-1 aggravated, the endotoxin-induced changes. 5. In conclusion, rats with genetic hypertension are more susceptible to endotoxaemia and this results in a greater extent of ALI compared with normotensive WKY rats.

Prolonged relaxation consistent with persistent soluble guanylyl cyclase activation in canine pulmonary artery following brief treatment with nitric oxide donors

Recent work has indicated that prolonged treatment with nitric oxide (NO) donors results in tissue storage of NO as S-nitrosothiols and N-nitrosamines. The possibility thus exists that NO treatment may result in the development of tissue stores of NO with functionally significant effects following removal of the original NO source. In these studies, the effects of 10 min treatment with two chemically distinct NO sources, S-nitrosoglutathione (GSNO) and ( Z)-1-( N, N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NO) were determined in canine pulmonary artery using a superfusion system that permitted continuous isometric force recording during addition and removal of the NO donors. Relaxation that persisted for up to 1 h after removal of the NO source, was demonstrated for both NO sources, but at lower concentrations relative to the relaxant EC 50 for GSNO versus DEA-NO. Persistent relaxation with both NO sources was fully reversed by both the sGC inhibitor, ODQ, and an inhibitor of cGMP-dependent protein kinase, Rp-8-Br-PET-cGMPS, indicating that persistent relaxation was consistent with persistent activation of the sGC-cGMP signaling pathway. In separate measurements, a GSNO-induced persistent increase in both tissue cGMP ([cGMP] i) and relaxation were fully reversed by both ODQ and the thiol reducing agent dithiothreitol (DTT). The results indicate that vascular smooth muscle is capable of converting short-lived NO responses following short term exposure to NO donors by a mechanism consistent with prolonged sGC activation, resulting in persistent relaxation. Reversal of this cGMP-dependent process with DTT suggests that it occurs via mechanisms that are thiol redox sensitive.

Downregulation of angiogenic factors and their downstream target molecules affects the deterioration of erectile function in a rat model of hypercholesterolemia

Objectives To evaluate how the expression of angiogenic factors and their downstream target molecules, which are potentially involved in penile homeostasis, is related to erectile dysfunction in a rat model of hypercholesterolemia. Methods Fifty-six 2-month-old male Sprague-Dawley rats were included in this study. The control animals (n = 28) were fed a normal diet, and the experimental animals (n = 28) were fed a diet containing 4% cholesterol and 1% cholic acid for 3 months. Erectile function was evaluated by cavernous nerve electrical stimulation, and cavernous tissue was harvested for histologic examination (n = 12, respectively). Cavernous tissue specimens from the remaining rats were used for reverse transcriptase–polymerase chain reaction (RT-PCR), Western blot, or cyclic guanosine monophosphate (cGMP) measurement. Results The ratio of maximal intracavernous pressure to mean arterial pressure was significantly lower in the hypercholesterolemic rats than in the controls ( P <0.01). Analysis by RT-PCR and Western blot showed significantly lower gene expression of vascular endothelial growth factor (VEGF), angiopoietin-1, and angiopoietin-2 and significantly lower protein expression of VEGF, angiopoietin-1, angiopoietin-2, the ratio of phospho-Akt to Akt, and phospho-endothelial nitric oxide synthase (eNOS) to eNOS in hypercholesterolemic rats than in controls. Cavernous tissue cGMP concentrations and endothelial area were also significantly lower in hypercholesterolemic rats than in controls ( P <0.01). Conclusions Downregulation of the expression of the angiogenic factors and their downstream signal molecules, and decreased endothelial content in the corpus cavernosum of hypercholesterolemic rats might play important roles in the deterioration of erectile function.

DOWN-REGULATION OF ANGIOGENIC FACTORS AND THEIR DOWNSTREAM TARGET MOLECULES AFFECT THE DETERIORATION OF ERECTILE FUNCTION IN A RAT MODEL OF HYPERCHOLESTEROLEMIA

To evaluate how the expression of angiogenic factors and their downstream target molecules, which are potentially involved in penile homeostasis, is related to erectile dysfunction in a rat model of hypercholesterolemia.Fifty-six 2-month-old male Sprague-Dawley rats were included in this study. The control animals (n = 28) were fed a normal diet, and the experimental animals (n = 28) were fed a diet containing 4% cholesterol and 1% cholic acid for 3 months. Erectile function was evaluated by cavernous nerve electrical stimulation, and cavernous tissue was harvested for histologic examination (n = 12, respectively). Cavernous tissue specimens from the remaining rats were used for reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, or cyclic guanosine monophosphate (cGMP) measurement.The ratio of maximal intracavernous pressure to mean arterial pressure was significantly lower in the hypercholesterolemic rats than in the controls (P <0.01). Analysis by RT-PCR and Western blot showed significantly lower gene expression of vascular endothelial growth factor (VEGF), angiopoietin-1, and angiopoietin-2 and significantly lower protein expression of VEGF, angiopoietin-1, angiopoietin-2, the ratio of phospho-Akt to Akt, and phospho-endothelial nitric oxide synthase (eNOS) to eNOS in hypercholesterolemic rats than in controls. Cavernous tissue cGMP concentrations and endothelial area were also significantly lower in hypercholesterolemic rats than in controls (P <0.01).Downregulation of the expression of the angiogenic factors and their downstream signal molecules, and decreased endothelial content in the corpus cavernosum of hypercholesterolemic rats might play important roles in the deterioration of erectile function.

The FGF system positively regulates vascular integrity after ischemic injury

Background The fibroblast growth factor (FGF) system plays significant roles in a wide variety of physiologic and pathologic conditions; however, early embryonic lethality of mice lacking either Fgfr-1 or Fgfr-2 precludes further studies focusing on FGF functions in the post-developmental stage. Methods We generated a conditional transgenic mouse line that expresses truncated dominant-negative FGFR1 (FGF-R1DN) in the endothelium under the control of tetracycline regulated Tie2 promoter. The expression of FGF-R1DN was induced in ten-week old mice, and four weeks later hindlimb ischemia was produced by ligation of the right femoral artery. Results FGF-R1DN mice demonstrated extensive toe/foot loss on the ischemic side of the leg following femoral artery ligation, accompanied by extensive apoptosis and concomitant reduction in tissue cGMP levels compared to control mice. Micro-CT and stereo-microscope analyses demonstrated vascular leakage and significant retention of contrast reagent throughout the ischemic region at the early stage of ischemic injury in FGF-R1DN mice. Introduction of FGF-R1DN adenovirus in rat femoral artery showed rearrangement of endothelial cell and disrupted cell junctions. Moreover, inhibition of FGF signaling in bovine aortic endothelial cells also showed displacement of VE-cadherin and ZO-1 from cell junctions, suggesting that the FGF signaling is required for the formation of endothelial junctions. Conclusion The FGF system appears to control maintenance of endothelial barrier function and vascular integrity, which plays an important role for tissue recovery from ischemic injury.

Nitric Oxide-Dependent Reduction in Soluble Guanylate Cyclase Functionality Accounts for Early Lipopolysaccharide-Induced Changes in Vascular Reactivity

We investigated the role of soluble guanylate cyclase in lipopolysaccharide-induced hyporesponsiveness to phenylephrine. The effects of phenylephrine on the blood pressure of female Wistar rats were evaluated at 2, 8, and 24 h after lipopolysaccharide injection (12.5 mg/kg i.p.). Vasoconstrictive responses to phenylephrine were reduced 40 to 50% in all time periods. Methylene blue, a soluble guanylate cyclase inhibitor (15 micromol/kg i.v.) restored the reactivity to phenylephrine in animals injected with lipopolysaccharide 2 and 24 h earlier. However, it failed to do so in animals injected with lipopolysaccharide 8 h earlier. Incubation with sodium nitroprusside (SNP) increased lung and aorta cGMP levels in control animals and in tissues of rats treated with lipopolysaccharide 24 h earlier. However, SNP failed to increase tissue cGMP in rats injected 8 h earlier. Lipopolysaccharide reduced the vasodilatory response to NO donors 8 h after injection. This effect and the decreased lung cGMP accumulation in response to SNP were reversed by an NO synthase blocker. Guanylate cyclase protein levels were lower than controls in lungs harvested from rats injected 8 h earlier and were back to normal values in lungs of rats injected 24 h earlier with lipopolysaccharide. Thus, data indicate that there is a temporal window of 8 h after lipopolysaccharide injection in which soluble guanylate cyclase is not functional and that this loss of function is NO-dependent. Thus, the putative use of soluble guanylate cyclase inhibitors in the treatment of endotoxemia may be beneficial mainly at early stages of this condition.

Nitric oxide sensitivity in pulmonary artery and airway smooth muscle: a possible role for cGMP responsiveness

We aimed to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Porcine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used in concentration-response studies to the NO donor (Z)-1-[N-2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM consistently exhibited greater relaxation at a given DETA-NO concentration (NO responsiveness) than TSM NO responsiveness, with DETA-NO log EC(50) being -6.55 +/- 0.11 and -5.37 +/- 0.13 for PASM and TSM, respectively (P < 0.01). We determined relationships between tissue cGMP concentration ([cGMP](i)) and relaxation using the particulate guanylyl cyclase agonist atrial natriuretic peptide. Atrial natriuretic peptide resulted in nearly complete relaxation, with no detectable increase in [cGMP](i) in PASM and only 20% relaxation (10-fold increase in [cGMP](i)) in TSM, indicating that TSM is less cGMP responsive than PASM. Total cGMP-dependent protein kinase I (cGKI) mRNA expression was greater in PASM than in TSM (2.23 +/- 0.36 vs. 0.93 +/- 0.31 amol mRNA/mug total RNA, respectively; P < 0.01), but total cGKI protein expression was not significantly different (0.56 +/- 0.07 and 0.49 +/- 0.04 ng cGKI/mug protein, respectively). The phosphotransferase assay for the soluble fraction of tissue homogenates demonstrated no difference in the cGMP EC(50) between PASM and TSM. The maximal phosphotransferase activity indexed to the amount of total cGKI in the homogenate differed significantly between PASM and TSM (1.61 +/- 0.15 and 1.04 +/- pmol.min(-1).ng cGKI(-1), respectively; P < 0.05), suggesting that cGKI may be regulated differently in the two tissues. A novel intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is thus greater cGMP responsiveness from increased cGKI-specific activity in PASM.

Anandamide elicits an acute release of nitric oxide through endothelial TRPV1 receptor activation in the rat arterial mesenteric bed

In the isolated rat mesenteric bed, the 1 min perfusion with 100 nm anandamide, a concentration that did not evoke vasorelaxation, elicited an acute release of 165.1 +/- 9.2 pmol nitric oxide (NO) that was paralleled by a 2-fold increase in cGMP tissue levels. The rise in NO released was mimicked by either (R)-(+)-methanandamide or the vanilloid receptor agonists resiniferatoxin and (E)-capsaicin but not by its inactive cis-isomer (Z)-capsaicin. The NO release elicited by either anandamide or capsaicin was reduced by the TRPV1 receptor antagonists 5'-iodoresiniferatoxin, SB 366791 and capsazepine as well as by the cannabinoid CB(1) receptor antagonists SR 141716A or AM251. The outflow of NO elicited by anandamide and capsaicin was also reduced by endothelium removal or NO synthase inhibition, suggesting the specific participation of endothelial TRPV1 receptors, rather than the novel endothelial TRPV4 receptors. Consistently, RT-PCR showed the expression of the mRNA coding for the rat TRPV1 receptor in the endothelial cell layer, in addition to its expression in sensory nerves. The participation of sensory nerves on the release of NO was precluded on the basis that neonatal denervation of the myenteric plexus sensory nerves did not modify the pattern of NO release induced by anandamide and capsaicin. We propose that low concentrations of anandamide, devoid of vasorelaxing effects, elicit an acute release of NO mediated predominantly by the activation of endothelial TRPV1 receptors whose physiological significance remains elusive.

Probucol Protects against Hypochlorite-induced Endothelial Dysfunction: IDENTIFICATION OF A NOVEL PATHWAY OF PROBUCOL OXIDATION TO A BIOLOGICALLY ACTIVE INTERMEDIATE

Atherosclerosis is associated with endothelial dysfunction and a heightened state of inflammation characterized, in part, by an increase in vascular myeloperoxidase and proteins modified by its principal oxidant, hypochlorous acid (HOCl). Here we examined whether probucol could protect against endothelial dysfunction induced by the two-electron oxidant HOCl. Hypochlorous acid eliminated endothelium-dependent relaxation of rabbit aorta, whereas endothelial function and tissue cGMP was preserved and elevated, respectively, in animals pretreated with probucol. Exogenously added probucol also protected against HOCl-induced endothelial dysfunction. In vitro, HOCl oxidized probucol in a two-phase process with rate constants k(1) = 2.7 +/- 0.3 x 10(2) and k(2) = 0.7 +/- 0.2 x 10(2) m(-1) s(-1) that resulted in a dose- and time-dependent accumulation of probucol-derived disulfoxide, 4,4'-dithiobis(2,6-di-tert-butyl-phenol) (DTBP), DTBP-derived thiosulfonate, disulfone, and sulfonic acid, together with 3,3',5,5'-tetra-tert-butyl-4,4'-diphenoquinone (DPQ) as determined by high performance liquid chromatography and mass spectrometry. Like HOCl, selected one-electron oxidants converted probucol into DTBP and DPQ. Also, dietary and in vitro added DTBP protected aortic rings from HOCl-induced endothelial dysfunction and in vitro oxidation by HOCl gave rise to the thiosulfonate, disulfone, and sulfonic acid intermediates and DPQ. However, the product profiles of the in vitro oxidation systems were different from those in aortas of rabbits receiving dietary probucol or DTBP +/- HOCl treatment. Together, the results show that both probucol and DTBP react with HOCl and protect against HOCl-induced endothelial dysfunction, although direct scavenging of HOCl is unlikely to be responsible for the vascular protection by the two compounds.

Effects of Nitric Oxide Synthase and Heme Oxygenase Inducers and Inhibitors on Molecular Signaling of Erectile Function

Changes in gene expression and the enzymatic activity of inducible nitric oxide synthase (iNOS), heme oxygenase isoenzymes 1 and 2 (HO-1 and HO-2) and cyclic GMP (cGMP) tissue levels in the corpora cavernosa (CC) were investigated experimentally in sixty Sprague-Dawley rats subjected to either HO or NOS inducers or inhibitors. They were divided into 5 groups (n = 12/group): Control group, L-arginine group which received L-arginine as NOS inducer, L-nitroarginine methyl ester (L-NAME) group which received L-NAME as NOS inhibitor, Hemin group which received hemin as HO inducer and Zn protoporphyrin (ZnPP) group which received ZnPP as HO inhibitor. The results of this study demonstrated that there was co-induction as well as co-repression of iNOS and HO-1 gene expression using either the inducers or inhibitors of both enzymes, whereas HO-2 gene expression was found to be upregulated by the use of L-arginine only in L-arginine rat group. HO-2 gene expression was not changed by the use of either HO inducer, suppressor or NOS suppressor. cGMP tissue levels in CC was significantly elevated in rats receiving HO inducer (hemin) as well as in those receiving NOS inducer (arginine) as compared with the control group, with a significant elevation in cGMP levels in hemin group as compared with L-arginine group. The use of hemin appears to be more efficient and dominates the use of arginine in inducing HO activity as well as cGMP tissue levels. These data indicate that HO and its product carbon monoxide (CO) are supervising nitric oxide as a signaling molecule in erectile function. On the other hand, There was a significant decrease in cGMP levels in HO inhibitor and in NOS inhibitor groups with a significant decrease in its levels in ZnPP group as compared with L-NAME group. HO enzymatic activity was significantly elevated in the hemin group, whereas it was significantly decreased in ZnPP and in arginine rat groups in comparison with the other groups. NOS activity was significantly elevated in arginine group and in ZnPP group and it was significantly decreased in L-NAME and in hemin rat groups in comparison with the other rat groups. Conclusion: The use of either NOS or HO inducers can equally enhance erectile function via upregulation of gene expression of the two signaling genes involved in erection as well as through upregulation of the tissue levels of cGMP, the mediator of vasodilatation. Our data indicate that HO/CO system is supervising and dominating nitric oxide as a signaling molecule in erectile function. Thus, induction of HO may have therapeutic implications for management of erectile dysfunction.

Cavernosal dysfunction in a rabbit model of hyperhomocysteinaemia

To investigate the effect of sustained hyperhomocysteinaemia (HHCy) on cavernosal smooth muscle function in a rabbit model of HHCy, developed using a methionine-enriched diet in which cavernosal responses were characterized, as elevated plasma levels of homocysteine may be a risk factor for vasculogenic erectile dysfunction. Six New Zealand White rabbits were fed a diet supplemented with methionine (20 g/kg chow) for 4 weeks, while six control animals were fed a standard diet. Cavernosal strips were mounted in an organ bath and relaxation assessed when stimulated with carbachol, sodium nitroprusside (SNP), or noncholinergic, nonadrenergic (NANC)-mediated relaxation to electrical-field stimulation (EFS). Cavernosal tissue cGMP levels were assessed using an enzyme-linked immunosorbent assay, and superoxide (O(2) (.-)) production assessed using an assay of the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c. The methionine-rich diet led to an early but sustained HHCy; cavernosal strips from animals after 4 weeks of HHCy had a significantly impaired relaxation response to carbachol, an index of endothelium-dependent nitric oxide (NO)-mediated relaxation. This impairment was reversed by incubating with either SOD or catalase. Relaxation with either SNP, an index of endothelium-independent NO-mediated relaxation, or NANC-mediated EFS-induced relaxation, was unaffected by HHCy. There was a corresponding significant reduction in cavernosal cGMP levels (index of NO activity) in the HHCy group, with a more than five-fold increase in cavernosal tissue O(2) (.-) production. Supplementing the diet of rabbits with methionine for 4 weeks caused an early and sustained HHCy and promoted a marked inhibitory effect on endothelium-dependent relaxation and NO formation in isolated corpus cavernosum, an effect mediated by reactive oxygen species.

Involvement of a nitric oxide-cyclic guanosine monophosphate pathway in control of Fallopian tube contractility

Fallopian tube smooth muscle contractions are physiologically related to transport of the ovum within the oviduct. Nitric oxide (NO) has proved to be a mediator of tubal contractility. The main pathway by which NO exerts its relaxing effect on tubal contractions has not been fully elucidated. NO-mediated effects may be cyclic guanosine monophosphate (cGMP)-dependent or cGMP-independent. The objective of the present study was to investigate whether a NO-cGMP pathway is present in the Fallopian tube and, if present, to examine whether this pathway is involved in tubal contractility. Tubal smooth muscle strips were mounted in organ baths for measurement of tissue cGMP and for isometric recording of contractile activity. Following administration of the NO donor spermine NONOate a more than three-fold increase in tissue levels of cGMP was measured. Pretreatment with inhibitors of cGMP production prior to administration of spermine NONOate resulted in similar levels of cGMP as found in strips exposed to only plain buffer solution. Administration of spermine NONOate to muscle baths resulted in a significant inhibition of contractile activity, while pretreatment with inhibitors of cGMP production almost eliminated the relaxing effect of the NO donor. This study showed that a NO-cGMP pathway is present in the Fallopian tube and that the pathway is involved in Fallopian tube contractility.

Early preconditioning prevents the loss of endothelial nitric oxide synthase and enhances its activity in the ischemic/reperfused rat heart

Cardiac ischemia may be responsible for either the loss of endothelial nitric oxide synthase (eNOS) or changes in its activity, both conditions leading to coronary dysfunction. We investigated whether early ischemic preconditioning was able to preserve eNOS protein expression and function in the ischemic/reperfused myocardium. Langendorff-perfused rat hearts were subjected to 20 min global ischemia, followed by 30 min reperfusion (I/R). A second group of hearts was treated as I/R, but preconditioned with three cycles of 5 min-ischemia/5 min-reperfusion (IP). Cardiac contractility markedly decreased in I/R, consistently with the rise of creatine kinase (CK) activity in the coronary effluent, whilst ischemic preconditioning significantly improved all functional parameters and reduced the release of CK. Western blot analysis revealed that the amount of eNOS protein decreased by 54.2% in I/R with respect to control (p < 0.01). On the other hand, NOS activity was not significantly reduced in I/R, as well as cGMP tissue levels, suggesting that a parallel compensatory stimulation of this enzymatic activity occurred during ischemia/reperfusion. Ischemic preconditioning completely prevented the loss of eNOS. Moreover, both NOS activity and cGMP tissue level were significantly higher (p < 0.05) in IP (12.7 ± 0.93 pmol/min/mg prot and 58.1 ± 12.2 fmol/mg prot, respectively) than I/R (7.34 ± 2.01 pmol/min/mg prot and 21.4 ± 4.13 fmol/mg prot, respectively). This suggest that early ischemic preconditioning may be useful to accelerate the complete recovery of endothelial function by preserving the level of cardiac eNOS and stimulating the basal production of nitric oxide.

Important role of nitric oxide in the effect of angiotensin-converting enzyme inhibitor imidapril on vascular injury.

To examine the possible role of the bradykinin-NO system in the action of ACE inhibitors, we studied the effects of imidapril, an ACE inhibitor, on inflammatory vascular injury by using AT1a-receptor-deficient (AT1aKO) mice. A polyethylene cuff was placed around the femoral artery of AT1aKO mice and wild-type (WT; C57BL/6J) mice. Neointimal area in cross sections of the artery was measured 14 days after cuff placement. A low dose of imidapril (1 mg/kg per day), which did not affect blood pressure, was administered by gavage. Expression of monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)-alpha was detected by immunohistochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR) 7 days after the operation. Neointimal formation, vascular smooth muscle cell proliferation, and expression of MCP-1 and TNF-alpha were attenuated in the injured artery in AT1aKO mice compared with those in WT mice. Imidapril inhibited neointimal formation, DNA synthesis of vascular smooth muscle cells, and expression of MCP-1 and TNF-alpha in AT1aKO mice as well as in WT mice. In addition, imidapril increased tissue cGMP content after cuff placement. These inhibitory effects of imidapril were significantly reduced or abolished by a bradykinin receptor antagonist, Hoechst 140, or an NO synthase inhibitor, L-NAME, both in WT and AT1aKO mice. Treatment with imidapril did not change AT2 receptor and ACE expression detected by RT-PCR in the injured artery. These results indicate that not only blockade of angiotensin II production but also activation of the bradykinin-NO system plays an important role in the beneficial effects of imidapril on vascular remodeling.